(A) Non-CT and CT constructs were designed using the Tol2 transgenesis system with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription of the mCherry open reading frame (see Supplement for maps and sequence information). All constructs harbored heart-specific GFP (cmlc2-GFP) which enabled identification of transgenic embryos. (B) Widespread expression of mCherry was observed in non-CT embryos (386 out of 386 transgenic embryos), whereas near complete loss of mCherry was observed in CT-mCherry embryos at 2dpf (370 out of 402 transgenic embryos).

Convergent silencing of the zebrafish One Eyed Pinhead (OEP) gene with the β-actin promoter driving sense transcription and an inverted CMV promoter driving antisense transcription. The OEP open reading frame was directly cloned between the two promoters. (B–G) Compared to uninjected control embryos (UICs), CT-OEP injected embryos phenocopied OEP mutants with curved body axis, cyclopia (black arrow in C), and reduced notochord (red arrows in F compared to G). In 12% of the CT-OEP embryos, we observed a more severe phenotype with complete loss of eye development (red asterisk in E). (H) Graph showing % transient transgenics showing WT, classic OEP, or more severe phenotypes. n = 167. (I). Silencing of OEP is Dicer dependent. Embryos were injected as indicated in the absence or presence of Dicer MO or a control mismatch morpholino. **p < 0.005.

(A) Convergent silencing of the miR-27a/b genes with ubiquitin promoters driving both sense and antisense transcription. A synthetic DNA sequence encompassing precursor sequences for both miR-27a and miR-27b were directly cloned between the two promoters. (B) Compared to control embryos injected with dye only (DIC), lateral and ventral views show that CT-miR-27a/b injected embryos phenocopy miR-27a/b morphants (see Supplemental Figure 4) with defects in pharyngeal arch morphogenesis, craniofacial defects, and inhibition of pectoral fin outgrowth as shown by decreased staining of ECM (cartilage) with alcian blue. (C) Compared to DICs, craniofacial defects observed with alcian blue staining were observed in 8–9.5% of CT-miR-27a/b embryos using two different plasmid clones of the construct in (A). n = 191 for DIC, n = 458 for CT-miR-27a/b clone 1, and n = 272 for CT-miR-27a/b clone 2. All images are at 4dpf.

miR-27a/b morphants.

Uninjected (UIC) embryos or embryos injected with morpholinos against miR- 27a/b were stained with alcian blue at 4dpf to detect extracellular matrix and developing cartilage.